Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
Stem cells are undifferentiated cells which are capable of proliferation and, self-maintenance, have the ability and capacity to generate a large repertoire of functional, differentiated progeny and are capable of responding to stimuli to initiate a regeneration of tissue after an injury. During embrogenesis, and after birth, stem cells are present in tissues which have high turnover of terminally differentiated cells such as blood and skin cells. Stem cells are also found to exist in tissues with little turnover of differentiated cells. An example mammalian tissue exhibiting low turnover is the adult mammalian central nervous system or central nervous system (CNS) [Reynolds, B. A. and Weiss, S., Science 255: 1707-1710, 1992; Richards et al., Natl. Acad. Sci. USA 89: 8591-8595, 1992; McKay, R., Science 276: 66-71, 1997; Gage, F. H., Science 287: 1433-1438, 2000].
The putative role of stem cells in the adult animal or human is to replace cells which are lost by natural cell death, injury or disease. To date, treatment for replacing defective or damaged organs or tissues has primarily been achieved via allogenic transplantation and by the administration of pharmaceutical compounds to reduce an immune response which can arise when allographed material is incompatible with the recipient. Recently, the concept of tissue grafting has been applied to the treatment of numerous diseases. In allografting procedures, either whole organs may be replaced or transplanted or small sections of tissue replaced such as in a skin graft. More recently, multipotent stem cells have been proposed for use in allograft procedures. In this type of allograft strategy, stem cells which have the capacity to differentiate into a particular tissue type are transplanted into a recipient.
Generally, stem cells then receive signals from surrounding tissue or other forms of stimulus resulting ultimately hi differentiation into a mature cell. In such procedures, it is necessary to isolate stem cells with the potential to develop along a particular pathway and into a desired mature cell. In order to achieve this it is necessary to isolate a quantity of stem cells. Generally, large numbers of stem cells are required. Suitable sources of sufficient numbers of stem cells can be harvested from embryonic and/or adult sources. However, current protocols for the culture of stem cells are cumbersome and often cannot provide sufficient numbers of stem cells required for tissue replacement therapy. In fact, hemopoietic stem cells cannot be expanded in culture. NSCs, however, do have the capacity to be expanded in culture but, until the advent of the present invention, it was not possible to initiate the culture with a homogenous population of NSCs. Thus, the development of stem cell tissue replacement therapy will require an ability to isolate and culture appropriate quantities of stem cells that will differentiate along a known pathway.
The fertilized egg is the ultimate stem cell from which all other cell lineages derive. As development proceeds, early embryonic cells respond to growth and differentiation signals which gradually narrow the cell's developmental potential, until the cells reach developmental maturity, and become terminally differentiated. These terminally differentiated cells have specialized functions and characteristics, and represent the last step in a multi-step process of precursor cell differentiation into a particular cell. The transition from one step to the next in cell differentiation is governed by specific biochemical mechanisms that gradually control the progression of cell differentiation until maturity is reached. It is clear that differentiation of tissues and cells is a gradual process that follows specific steps until a terminally differentiated state is reached. Thus, stem cells represent a source of undifferentiated cells which can be used in the maintenance of tissues whether during normal life or in response to injury and disease.
An expanded pool of stem and progenitor cells, as well as non-terminally differentiated cells that could supply a desired differentiation phenotype would be of great value in therapies that involve tissue replacement. To allow cell replacement therapy to become widely applicable in the clinical domain, a considerable challenge is to address the problem of provision of suitable quantities of replenishing stem cells that are capable of differentiating into a particular cell type.
During development of the CNS, multipotent precursor cells, i.e. NSCs, proliferate, giving rise to transiently dividing progenitor cells that eventually differentiate into the cell types that compose the adult brain. Some NSCs have also been shown to possess the potential to give rise to hemopoietic and muscle cells (Clark, D. L., Science 288: 1660-1663, 2000; Bjornson et al., Science 283: 534-537, 1999). NSCs are classically defined as having the ability to self-renew, to proliferate and been shown to differentiate into multiple different phenotypic lineages including neurons, astrocytes and oligodendrocytes. NSC activity has been detected from all mammalian species studied to date including mice, rats, non-human primates and humans.
Although methods for obtaining and culturing multipotent NSCs have been previously described, a major problem with the resulting population of cells is that they provide very low percentages of NSCs. Importantly, the populations of cells obtained are mixed and contain other cell types. Such cell populations would need to undergo complicated enrichment to increase the proportion of NSCs if used for transplantation procedures. Such cultivation procedures, however, do not address the presence of heterogeneity in the mixed cells in the populations which have diverse differentiation characteristics. Typically, less that 0.1% of cells obtained from neural tissue are multipotent stem cells. Thus, there is a clear need for a method which permits the purification of these multipotent stem cells to homogeneity.
In work leading up to the present invention, the inventors sought to provide a method for the isolation of a substantially homogenous population of undifferentiated cells and in particular stem cells and more particularly NSCs. The inventors utilized cell surface markers in combination with cell sorting procedures based on cell size to purify a population of NSCs to substantial homogeneity. Surprisingly, the inventors determined that a highly homogenous population of NSCs can be isolated from mammalian brain tissue. Furthermore, the inventors have developed protocols and methods to propagate NSCs and form a highly homogenous population of NSCs which retain the ability to differentiate into mature cell lineages. Thus, the instant invention overcomes the difficulty of producing undifferentiated cells in purified form. The homogeneous cell populations of the present invention are useful in a range of situations including transplantation methodologies in therapeutic approaches to repair and replace damaged and/or dysfunctional tissue, tissue augmentation, delivery of genetic and proteinaceous molecules including therapeutic cytokine delivery, identification of factors controlling differentiation and/or proliferation including inhibitors thereof and identification of diagnostic and therapeutic markers. Furthermore, by identifying and purifying, a NSC population, it can be determined which factor(s) regulate the proliferation, self-maintenance and differentiative properties of NSCs in situ, thereby overcoming the need for transplantation of donor cells. The activation of NSCs in situ represents a major advantage over transplantation therapies in that it avoids major surgery and issues of tissue rejection.